Methods, kits, and devices utilizing HTR2C polymorphisms for diagnosing, prognosing, and treating psychiatric disorders in a patient

ABSTRACT

Disclosed are methods, kits, and devices for diagnosing and treating psychiatric, disorders and the symptoms thereof. The methods, kits, and devices relate to identifying genetic markers that may be utilized to diagnose and/or prognose a patient and treat the diagnosed and/or prognosed patient by administering a drug the patient based on the genetic marker having been identified. Genetic markers identified in the methods may include HTR2C polymorphisms such as a polymorphism resulting in a Cys23Ser amino acid substitution, an rs3813929 (−759C/T) polymorphism, and an rs518147 (−697G/C) polymorphism).

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62/043,937, filed onAug. 29, 2014, the content of which is incorporated herein by referencein its entirety.

BACKGROUND

The invention relates to methods for diagnosing and treating psychiatricdisorders. In particular, the intention relates to methods foridentifying genetic markers that are associated with treatment responsefor psychiatric disorders in a patient and administering, drugs to thepatient based on identifying the genetic markers. The genetic markersmay include polymorphisms such as single nucleotide polymorphisms (SNPs)and the drugs may include typical and atypical antipsychotic drugs(APDs).

SUMMARY

Disclosed are methods, kits, and devices for diagnosing and treatingpsychiatric disorders and the symptoms thereof. The methods, kits, anddevices relate to identifying genetic markers that may be utilized todiagnose and/or prognose a patient and treat the diagnosed and/orprognosed patient by administering a drug to the patient based on thegenetic marker having been identified. Genetic markers identified in themethods may include HTR2C polymorphisms. Based on the polymorphism beingidentified in the patient, the patient may be identified as havingresponsiveness to an antipsychotic drug (APD), such as a typical APD oran atypical APD. As such, the patient may be treated by administeringthe APD to treat the psychiatric disorder and/or the symptoms thereofafter the HTR2C polymorphism has been identified.

In some embodiments, the disclosed methods related to methods fortreating a psychiatric disorder or the symptoms thereof in a patient.The disclosed methods may comprise the following steps: (a) determiningwhether the patient has a HTR2C polymorphism, or receiving the resultsof test indicating that a patient has a HTR2C polymorphism; and (b)administering an antipsychotic drug (APD) if the patient has the HTR2Cpolymorphism. In the disclosed methods, the psychiatric disorder mayinclude, but is not limited to schizophrenia (e.g., schizophreniacharacterized by positive symptoms, negative symptoms, and/or cognitivesymptoms), bipolar disorder, and psychiatric depression with psychoticfeatures. Suitable HTR2C polymorphisms detected in the methods orindicated in the test results utilized in the methods may include, butare not limited to a polymorphism resulting in a Cys23Ser amino acidsubstitution e.g., a. UGC←→UCC transversion or a UGU←→UCU transversion)an rs3813929 (−759C/T) polymorphism, and an rs518147 (−697G/C)polymorphism.

In some embodiments of the disclosed methods, the patient may haveundergone treatment prior to the disclosed methods being performed andthe patient may have been diagnosed with a treatment resistantpsychiatric disorder prior to the method being performed. Accordingly,the methods contemplated herein include methods for determiningtreatment responsiveness and treating patients with the appropriate APD.

The presently disclosed methods relate to HTR2C polymorphisms, which mayinclude but are not limited to a polymorphism resulting in a. Cys23Seramino acid substitution (e.g., a UGC←→UCC transversion or a UGU←→UCUtransversion), an rs3813929 (−759C/T) polymorphism, and an rs518147(−697G/C) polymorphism. The disclosed methods may include determining ordetecting a nucleotide sequence associated with HTR2C polymorphisms. Insome embodiments, the methods including determining whether the patienthas one or more HTR2C polymorphisms by sequencing, a nucleic acid sampleobtained from the patient. In other embodiments, the methods may includedetermining whether the patient has one ore more HTR2C polymorphisms bytreating a nucleic acid sample obtained from the patient with a nucleicacid probe (e.g. a probe that hybridizes specifically to a nucleic acidsequence comprising the HTR2C polymorphic allele). The methods mayinclude determining whether the patient is homozygous or heterozygousfor a HTR2C polymorphic allele. Further, the methods may includeadministering a pharmaceutical agent if the patient is found to behomozygous or heterozygous for the HTR2C polymorphic allele.

The disclosed methods typically include treating a patient based on thegenotype of the patient with respect to the HTR2C polymorphism, whichmay include but are not limited to a polymorphism resulting in aCys23Ser amino acid substitution, an rs3813929 (−759C/T) polymorphism,and an rs518147 (−697G/C) polymorphism. For example, the methodstypically include administering a pharmaceutical agent to the patient ifthe patient is homozygous or heterozygous for a HTR2C polymorphicallele.

Also disclosed herein are kits and devices for performing the disclosedmethods, and systems comprising the disclosed kits and devices. Forexample, the disclosed kits and devices may include and/or utilizereagents for diagnosing, prognosing, and/or treating a psychiatricdisease or disorder or the symptoms thereof in a patient. The presentlydisclosed kits and devices may include and/or utilize reagents such as:(a) reagents for detecting the genotype of a patient in regard to aHTR2C polymorphism (e.g., where the patient has a polymorphism resultingin a Cys23Ser amino acid substitution, where the patient is homozygousor heterozygous for the rs3813929 (−759C/T) polymorphic allele, and/orwhere the patient is homozygous or heterozygous for the rs518147(−697G/C) polymorphic allele). The kits and devices may include and/orutilize reagents for amplifying and or sequencing nucleic acidcomprising one or more HTR2C polymorphisms and/or reagents for probingnucleic acid comprising one or more HTR2C polymorphisms; and optionally(b) a pharmaceutical agent comprising an atypical drug for treating apsychiatric disease or disorder (e.g., clozapine olanzapine,risperidone, and sertindole). The reagents in the kit may includenucleic acid reagents (e.g., primers and/or probes that hybridize to theHTR2C gene and that may be utilized to amplify, sequence, and/or probethe HTR2C gene or an RNA expressed from the HTR2C gene) and non-nucleicacid reagents (e.g., polymerases and buffers). The pharmaceutical agentof the kits and devices may include a typical or atypical APD fortreating the psychiatric disease or disorder formulated foradministration to the patient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Haploview plot of SNPs in Caucasians from 1000 genome data.

FIG. 2. A meta-analysis of the Ser carrier for rs6318 (Cys23Ser)associated with treatment response (binary data) across six studies onCaucasian samples. A. Forest Plot. Experimental=Ser carrier;Control=Non-Ser carrier; Events=number of Responder; Total=number ofsubjects with that genotype. The squares represent odds ratio to have afavorable treatment response if it is over 1 and the horizontal linesshow the 95% confidence intervals of the corresponding odds ratio. ForVehof's study, the raw data was computed based on publishedimprovement/no improvement ratio, genotyping data, and odds ratio. BFunnel Plot is used to assesses publication bias in meta-analysisdatasets. If all studies come from a single population then the plotshould look like a funnel with the diameter of the funnel decreasing asthe sample size increases. In the absence of publication bias, oneshould expect a symmetrical funnel plot.

FIG. 3. Haploview plot of HTR2C SNPs in the tested sample and 1000genome sample stratified by ethnicity (European or African-American).

DETAILED DESCRIPTION

Disclosed are methods, kits, and devices for diagnosing and treatingpsychiatric disorders and the symptoms thereof. The methods, kits, anddevices are described herein using several definitions, as set forthbelow and throughout the application.

As used in this specification and the claims, the singular forms “a,”“an,” and “the” include plural forms unless the context clearly dictatesotherwise. For example, “a polymorphism” should be interpreted to mean“one or more polymorphisms” unless the context clearly dictatesotherwise. As used herein, the term “plurality” means “two or more.”

As used herein, “about”, “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of the term which are not clear to persons of ordinaryskill in the art given the context in which it is used, “about” and“approximately” will mean up to plus or minus 10% of the particular termand “substantially” and “significantly” will mean more than plus orminus 10% of the particular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising.” The terms “comprise”and “comprising” should be interpreted as being “open” transitionalterms that permit the inclusion of additional components further tothose components recited in the claims. The terms “consist” and“consisting of” should be interpreted as being “closed” transitionalterms that do not permit the inclusion of additional components otherthan the components recited in the claims. The term “consistingessentially of” should be interpreted to be partially closed andallowing the inclusion only of additional components that do notfundamentally alter the nature of the claimed subject matter.

The presently disclosed methods, kits, and devices relate to identifyinggenetic markers that may be utilized to diagnose and/or prognose apatient, and optionally treat the diagnosed and/or prognosed patient byadministering a drug, to the patient based on the genetic marker havingbeen identified.

As used herein, the term “patient,” which may be used interchangeablywith the terms “subject” or “individual,” refers to one who receivesmedical care, attention or treatment and may encompass a human patient.As used herein, the term “patient” is meant to encompass a person whohas a psychiatric disorder or is at risk for developing a psychiatricdisorder, which includes but is not limited to schizophrenia, bipolardisorder, and psychotic depression (e.g., depression with psychoticfeatures). For example, the term “patient” is meant to encompass aperson at risk for developing schizophrenia or a person diagnosed withschizophrenia (e.g., a person who may be symptomatic for schizophreniabut who has not yet been diagnosed). As used herein, “schizophrenia” mayinclude schizophrenia characterized by positive symptoms, negativesymptoms, cognitive symptoms, or any combination thereof. The term“patient” also is meant to encompass a person at risk for developingbipolar disorder or a person diagnosed with bipolar disorder (e.g., aperson who may be symptomatic for bipolar disorder but who has not yetbeen diagnosed). The term “patient” further is meant to encompass aperson at risk for developing depression (e.g., depression withpsychotic features). As such, the term “patient” further is meant toencompass a person at risk for developing depression with psychoticfeatures or a person diagnosed with depression with psychotic features(e.g., a person who may be symptomatic for depression with psychoticfeatures but who has not yet been diagnosed).

The disclosed methods may include: (a) detecting a HTR2C polymorphism ina nucleic, acid sample from a patient having a psychiatric disease ordisorder; and (h) administering an antipsychotic drug (APD) to thepatient after the HTRC2C polymorphism is detected. In some embodiments,the HTR2C polymorphism may be detected by a step that includesamplifying at least a portion of the HTR2C gene from the nucleic acidsample and detecting the HTR2C polymorphism in the amplified portion. Infurther embodiments, the HTR2C polymorphism may be detected by a stepthat includes sequencing at least a portion of the HTR2C gene from thenucleic acid sample or from art amplicon obtained by amplifying at leasta portion of the HTR2C gene from the nucleic acid sample. In evenfurther embodiments, the HTR2C polymorphism may be detected by a stepthat includes contacting nucleic acid comprising the HTR2C polymorphismwith a nucleic acid probe that hybridizes specifically to nucleic acidcomprising the HTR2C polymorphism.

Genetic markers identified in the methods typically include HTR2Cpolymorphisms. Exemplary HTR2C polymorphisms detected in the disclosedmethods may include, but are not limited to a polymorphism resulting ina Cys23Ser amino acid substitution, an rs3813929 (−759C/T) polymorphism,and an rs518147 (−697G/C) polymorphism. The disclosed methods mayinclude determining whether a patient is homozygous or heterozygous forthe HTR2C polymorphism (e.g., by determining whether a nucleic acidsample from the patient is homozygous or heterozygous for the HTR2Cpolymorphism). Methods, compositions, and kits for diagnosing,prognosing, and treating psychiatric disorders that include steps ofdetecting genetic polymorphisms also are disclosed in U.S. PublishedApplication No. 2015/0099741, the content of which is incorporate hereinby reference in its entirety.

As used herein, “HTR2C” refers to “5-hydroxytrptamine receptor 2Cisoform.” Gene information for HTR2C is provided at the NCBI database asfollows: “HTR2C 5-hydroxytryptamine (serotonin) receptor 2C, Gprotein-coupled [Homo sapiens (human)] Gene ID: 3358.” The HTR2C isoform“a precursor” is a 458 amino acid polypeptide, the amino acid sequenceof which is provided at the NCBI database under Accession No.NP_001243689 (SEQ ID NO:1). The HTR2C isoform “b precursor” is a 248amino acid polypeptide, the amino acid sequence of which is provided atthe NCBI database under Accession No. NP_001243690 (SEQ ID NO:2). Asused herein, a nucleic acid reagent that specifically hybridizes toHTR2C may include a nucleic acid reagent that specifically hybridizes toa polynucleotide that encodes the amino acid sequence of SEQ ID NO:1 orSEQ ID NO:2. For example, a nucleic acid reagent that specificallyhybridizes to HTR2C may include a nucleic acid reagent that specificallyhybridizes to the cDNA (or mRNA) encoding the HTR2C isoform “aprecursor” (see SEQ ID NO:3 and NCBI database CCDS59174)) or to the cDNA(or mRNA) encoding the HTR2C isoform “b precursor” (see SEQ ID NO:4 andNCBI database CCDS14564). A nucleic acid reagent that specificallyhybridizes to HTR2C may include a nucleic acid reagent that specificallyhybridizes to a portion of the HTR2C gene comprising a mutationresulting in a Cys23Ser mutation (e.g., a portion of SEQ ID NO:5 or SEQID NO:6). A nucleic acid reagent that specifically hybridizes to HTR2Cmay include a nucleic acid reagent that specifically hybridizes to aportion of the HTR2C gene comprising the rs3813929 SNP (e.g., a portionof SEQ ID NO:7 or 8). A nucleic acid reagent that specificallyhybridizes to HTR2C may include a nucleic acid reagent that specificallyhybridizes to a portion of the HTR2C gene comprising the rs3813929 SNP(e.g., a portion of SEQ ID NO:9 or 10).

The disclosed methods may include detecting a HTR2C polymorphism in anucleic acid sample from a patient having a psychiatric disease ordisorder. As used herein, a “psychiatric disease or disorder” mayinclude, but is not limited to schizophrenia, bipolar disorder, andpsychiatric depression.

Based on the HTR2C polymorphism being identified in the patient, thepatient may be identified as having responsiveness to an antipsychoticdrug (APD), such as a typical APD or an atypical APD. As such, thepatient may be treated by administering the API) to treat thepsychiatric disorder and/or the symptoms thereof after the HTR2Cpolymorphism has been identified.

Accordingly, the disclosed methods, kits, and devices optionally mayutilize or include an antipsychotic drug (APD). Suitable APDs mayinclude typical APDs and atypical APDs. APDs for use in the disclosedmethods, kits, and devices, may include, but are not limited toClozapine (Clozaril®), Benperidol (Anguil®, Benguil®, Frenactil®,Glianimon®), Bromperidol (Bromodol®, Impromen®), Droperidol (Droleptan®,Inapsine®), Haloperidol (Hatdol®, Serenace®), Moperone (Luvatren®),Pipamperone (Dipiperon®, Piperonil®), Timiperone (Celmanil®,Tolopelon®), Diphenylbutylpiperidine, Fluspirilene (Imap®), Penfluridol(Semap®), Pimozide (Orap®), Acepromazine (Plegicil®), Chlorpromazine(Largactil®, Thorazine®), Cyamemazine, (Tercian®), Dixyrazine (Esucos®),Fluphenazine (Modecate®, Permitil®, Prolixin®), Levomepromazine(Levinan®, Levoprome®, Nozinan®), Mesoridazine (Lidanil®, Serentil®),Perazine (Peragal®, Perazin®, Pernazinum®, Taxilan®), Pericyazine(Neulactil®, Neuleptil®), Perphenazine (Trilafon®), Pipotiazine(Lonseren®, Piportil®), Prochlorperazine (Compazine®), Promazine(Prozine®, Sparine®), Promethazine (Avomine®, Phenergan®), Prothipendyl(Dominal®), Thioproperazine (Majeptil®), Thioridazine (Aldazine®,Mellaril®, Melleril®), Trifluoperazine (Stelazine®), Trifinpromazine(Vesprin®), Chlorprothixene (Cloxan®, Taractan®, Truxal®), Clopenthixol(Sordinol®), Flupentixol (Depixol®, Fluanxol®), Tiotixene (Navane®,Thixit®), Zuclopenthixol (Acuphase®, Cisordinol®, Clopixol®), Clotiapine(Entumine®, Etomine®, Etumine®), Loxapine (Adasuve®, Loxitane®),Prothipendyl (Dominal®), Carpipramine (Defekton®, Prazinil®),Clocapramine (Clofekton®, Padrasen®), Molindone (Moban®), Mosapramine(Cremin®), Sulpiride (Meresa®), Sultopride (Barnetil®, Topral®),Veralipride (Agreal®), Amisulpride (Solian®), Amoxapine (Asendin®),Aripiprazole (Abilify®), Asenapine (Saphris®, Sycrest®), Blonanserin(Lonasen®), Iloperidone (Fanapt®, Fanapta®, Zomaril®), Lurasidone(Latuda®), Melperone (Buronil®, Buronon®, Eunerpan®, Melpax®, Neuril®),Olanzapine (Zyprexa®), Paliperidone (Invega®), Perospirone (Lullan®),Quetiapine (Seroquel®), Remoxipride (Roxiam®), Risperidone (Risperdal®),Sertindole (Serdolect®, Serlect®), Trimipramine (Surmontil®),Ziprasidone (Geodon®, Zeldox®), and Zotepine (Lodopin®, Losizopilon®,Nipolept®, Setous®). In some embodiments, the APD is an atypical APDthat is an inverse agonist of the HTR2C receptor (and optional that isan inverse agonist of the HTR2A receptor). For example, atypical APDsthat are inverse agonists of the HTR2C receptor and the HTR2A receptormay include clozapine, olanzapine, risperidone, and sertindole.

The disclosed methods, kits, and devices may utilize or include areagent that is utilized for detecting an HTR2C polymorphism. Suitablereagents may include nucleic acid reagents. For example, nucleic acidreagents may include reagents comprising a DNA oligonucleotide thathybridizes specifically to the HTR2C gene or that hybridizesspecifically to a polymorphism in the HTR2C gene. In some embodiments,the methods, kits, and device may utilize or include nucleic acidreagents that comprise one or more primers for sequencing at least aportion of the HTR2C gene (e.g., where the portion of the HTR2Ccomprises a HTR2C polymorphism selected from the group consisting of apolymorphism resulting in a Cys23Ser amino acid substitution, anrs3813929 (−759C/T) polymorphism, and an rs51814 (−697G/C)polymorphism). In further embodiments, the methods, kits, and device mayutilize or include nucleic acid reagents that comprise one or moreprimer pairs for amplifying at least a portion of the HTR2C gene (e.g.,where the portion of the HTR2C gene comprises a HTR2c polymorphismselected from the group consisting of a polymorphism resulting in aCys23Ser amino acid substitution, an rs3813929 (−759C/T) polymorphism,and an rs518147 (−697G/C) polymorphism).

The disclosed kits and/or devices disclosed herein may be assembled intosystems for performing the methods disclosed herein. Manual and/orautomated systems comprising the contemplated kits and/or devices arecontemplated herein.

As used herein the terms “diagnose” or “diagnosis” or “diagnosing” referto distinguishing or identifying a disease, syndrome or condition ordistinguishing or identifying a person having or at risk for developinga particular disease, syndrome or condition. As used herein the terms“prognose” or “prognosis” or “prognosing” refer to predicting an outcomeof a disease, syndrome or condition. The methods contemplated hereininclude diagnosing a psychiatric disorder in a patient that isassociated with a HTR2C polymorphism. The methods contemplated hereinalso include determining a prognosis for a patient having a psychiatricdisorder that is associated with a HTR2C polymorphism.

As used herein, the terms “treating” or “to treat” each mean toalleviate symptoms, eliminate the causation of resultant symptoms eitheron a temporary or permanent basis, and/or to prevent or slow theappearance or to reverse the progression or severity of resultantsymptoms of the named disease or disorder. As such, the methodsdisclosed herein encompass both therapeutic and prophylacticadministration. In particular, the methods contemplated herein includetreating a patient having or at risk for developing a psychiatricdisorder that is associated with a HTR2C polymorphism.

The present methods may include detecting a HTR2C polymorphism in apatient sample (e.g., a sample comprising nucleic acid). The term“sample” or “patient sample” is meant to include biological samples suchas tissues and bodily fluids. “Bodily fluids” may include, but are notlimited to, blood, serum, plasma, saliva, cerebral spinal fluid, pleuralfluid, tears, lactal duct fluid, lymph, sputum, and semen. A sample mayinclude nucleic acid, protein, or both.

The detected HTR2C polymorphism is present in nucleic acid. The term“nucleic acid” or “nucleic acid sequence” refers to an oligonucleotide,nucleotide or polynucleotide, and fragments or portions thereof, whichmay be single or double stranded, and represents the sense or antisensestrand. A nucleic acid may include DNA or RNA, and may be of natural orsynthetic origin. For example, a nucleic acid may include mRNA or cDNA.Nucleic acid may include nucleic acid that has been amplified (e.g.,using polymerase chain reaction). Nucleic acid may include genomicnucleic acid.

As used herein, the term “assay” or “assaying” means qualitative orquantitative analysis or testing.

As used herein the term “sequencing,” as in determining the sequence ofa polynucleotide, refers to methods that determine the base identity atmultiple base positions or that determine the base identity at a singleposition.

The term “amplification” or “amplifying” refers to the production ofadditional copies of a nucleic acid sequence. Amplification is generallycarried out using polymerase chain reaction (PCR) technologies known inthe art.

The term “oligonucleotide” is understood to be a molecule that has asequence of bases on a backbone comprised mainly of identical monomerunits at defined intervals. The bases are arranged on the backbone insuch a way that they can enter into a bond with a nucleic acid having asequence of bases that are complementary to the bases of theoligonucleotide. The most common oligonucleotides have a backbone ofsugar phosphate units. Oligonucleotides of the method which function asprimers or probes are generally at least about 10-15 nucleotides longand more preferably at least about 15 to 25 nucleotides long, althoughshorter or longer oligonucleotides may be used in the method. The exactsize will depend on many factors, which in turn depend on the ultimatefunction or use of the oligonucleotide. An oligonucleotide (e.g., aprobe or a primer) that is specific for a target nucleic acid will“hybridize” to the target nucleic acid under suitable conditions. Asused herein. “hybridization” or “hybridizing” refers to the process bywhich an oligonucleotide single strand anneals with a complementarystrand through base pairing under defined hybridization conditions.Oligonucleotides used as primers or probes for specifically amplifying(i.e., amplifying a particular target nucleic acid sequence) orspecifically detecting (i.e., detecting a particular target nucleic acidsequence) a target nucleic acid generally are capable of specificallyhybridizing, to the target nucleic acid.

The present methods and kits may utilize or contain primers, probes, orboth. The term “primer” refers to an oligonucleotide that hybridizes toa target nucleic acid and is capable of acting as a point of initiationof synthesis when placed under conditions in which primer extension isinitiated (e.g., printer extension associated with an application suchas PCR), For example, primers contemplated herein may hybridize to oneor more polynucleotide sequences comprising the HTR2C polymorphismsdisclosed herein. A “probe” refers to an oligonucleotide that interactswith a target nucleic acid via hybridization. A primer or probe may befully complementary to a target nucleic acid sequence or partiallycomplementary. The level of complementarity will depend on many factorsbased, in general, on the function of the primer or probe. For example,probes contemplated herein may hybridize to one or more polynucleotidesequences comprising the HTR2C polymorphisms disclosed herein. A primeror probe may specifically hybridize to a target nucleic acid (e.g.,hybridize under stringent conditions as discussed herein). Inparticular, primers and probes contemplated herein may hybridizespecifically to one or more polynucleotide sequences that comprise theHTR2C polymorphisms disclosed herein and may be utilized to distinguisha polynucleotide sequence comprising a minor allele from apolynucleotide sequence comprising the major allele.

An “oligonucleotide array” refers to a substrate comprising a pluralityof oligonucleotide primers or probes. The arrays contemplated herein maybe used to detect the HTR2C polymorphisms disclosed herein.

As used herein, the term “specific hybridization” indicates that twonucleic acid sequences share a high degree of complementarity. Specifichybridization complexes form under stringent annealing conditions andremain hybridized after any subsequent washing steps. Stringentconditions for annealing of nucleic acid sequences are routinelydeterminable by one of ordinary skill in the art and may occur, forexample, at 65° C. in the presence of about 6×SSC. Stringency ofhybridization may be expressed, in part, with reference to thetemperature under which the wash steps are carried out. Suchtemperatures are typically selected to be about 5° C. to 20° C. lowerthan the thermal melting, point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength and pH) at which 50% of the target sequence hybridizes toa perfectly matched probe. Equations for calculating Tm and conditionsfor nucleic acid hybridization are known in the art.

As used herein, a “target nucleic acid” refers to a nucleic acidmolecule containing a sequence that has at least partial complementaritywith a probe oligonucleotide, a primer oligonucleotide, or both, Aprimer or probe may specifically hybridize to a target nucleic acid.

The present methods may be performed to detect the presence or absenceof the disclosed HTR2C polymorphisms. Methods of determining thepresence or absence of a HTR2C polymorphism may include a variety ofsteps known in the art, including one or more of the following steps:reverse transcribing mRNA that comprises the HTR2C polymorphism to cDNA,amplifying nucleic acid that comprises the HTR2C polymorphism (e.g.,amplifying genomic DNA that comprises the HTR2C polymorphism),hybridizing a probe or a primer to nucleic acid that comprises the HTR2Cpolymorphisms (e.g., hybridizing a probe to mRNA, cDNA, or amplifiedgenomic DNA that comprises the HTR2C polymorphism), and sequencingnucleic acid that comprises the HTR2C polymorphism (e.g., sequencingcDNA, genomic DNA, or amplified DNA that comprises the HTR2Cpolymorphism.

A “polymorphism” refers to the occurrence of two or more alternativegenomic sequences or alleles between or among different genomes orindividuals, “Polymorphic” refers to the condition in which two or morevariants of a specific genomic sequence can be found in a population. A“polymorphic site” is the locus at which the variation occurs, A singlenucleotide polymorphism (SNP) is the replacement of one nucleotide byanother nucleotide at the polymorphic site. Deletion of a singlenucleotide or insertion of a single nucleotide also gives rise to singlenucleotide polymorphisms. “Single nucleotide polymorphism” preferablyrefers to a single nucleotide substitution. Typically, between differentindividuals, the polymorphic site can be occupied by two differentnucleotides which results in two different alleles with the most commonallele in the population (i.e., the ancestral allele) being referred toas the “major allele” and the less common allele in the population beingreferred to as the “minor allele.” An individual may be homozygous orheterozygous for the major allele or the minor allele of thepolymorphism. “Mutation” as utilized herein, is intended to encompass asingle nucleotide substitution, which may be recognized as a singlenucleotide polymorphism. Exemplary SNPs disclosed herein includers3813929 (−759C/T), and rs518147 (−697G/C).

In the methods and kits, the minor allele and/or the major alleleassociated with a polymorphism may be detected. The methods may includeand the kits and devices may be used for determining whether a patientis homozygous or heterozygous for a minor allele and/or major alleleassociated with a polymorphism (e.g., a SNP). The term “heterozygous”refers to having different alleles at one or more genetic loci inhomologous chromosome segments. As used herein “heterozygous” may alsorefer to a sample, a cell, a cell population or a patient in whichdifferent alleles (e.g., major or minor alleles of SNPs) at one or moregenetic loci may be detected. Heterozygous samples may also bedetermined via methods known in the art such as, for example, nucleicacid sequencing. For example, if a sequencing electropherogram shows twopeaks at a single locus and both peaks are roughly the same size, thesample may be characterized as heterozygous. Or, if one peak is smallerthan another, but is at least about 25% the size of the larger peak, thesample may be characterized as heterozygous. In some embodiments, thesmaller peak is at least about 15% of the larger peak. In otherembodiments, the smaller peak is at least about 10% of the larger peak.In other embodiments, the smaller peak is at least about 5% of thelarger peak. In other embodiments, a minimal amount of the smaller peakis detected.

As used herein, the term “homozygous” refers to having identical allelesmajor or minor alleles of SNPs) at one or more genetic loci inhomologous chromosome segments. “Homozygous” may also refer to a sample,a cell, a cell population, or a patient in which the same alleles at oneor more genetic loci may be detected. Homozygous samples may bedetermined via methods known in the art, such as, for example, nucleicacid sequencing. For example, if a sequencing electropherogram shows asingle peak at a particular locus, the sample may be termed “homozygous”with respect to that locus.

Suitable polymorphism for the presently disclosed, methods, kits, andarrays may include a polymorphism (or mutation) resulting in a Cys23Seramino acid substitution, an rs3813929 (−759C/T) polymorphism, and anrs518147 (−697G/C) polymorphism). For example, the SNP referred to bydbSNP reference ID No. rs3813929 is located on the X chromosome,position 114584047, which may be a thymidine (T) in the minor allele ora cytosine (C) in the major allele; and the SNP referred to by dbSNPreference ID No. rs518147 is located on the X chromosome, position114584109, which may be as cytosine (C) in the minor allele, or aguanine (G) in the major allele.

The present methods contemplate detecting a single nucleotidepolymorphism (SNP). For example, the present methods may detectrs3813929 in either one or both alleles of the patient. (See rs3813929SNP entry at the National Center for Biotechnology Information, whichentry is incorporated herein by reference and refers to a C←→Ttransition at the reference nucleotide position, where the C-allele isthe major allele and the T-allele is the minor allele). The presentmethods may detect a T-allele or a C-allele corresponding to thepolymorphism (i.e., a T-nucleotide or a C-nucleotide at the positionassociated with the rs3813929). The present methods may detect whether apatient is homozygous or heterozygous for a T-allele or C-allele (i.e.,whether the patient is TT, TC, or CC at the reference nucleotideposition for rs3813929).

The present methods also may detect rs518147 m either one or bothalleles of the patient. (See rs518147 SNP entry at the National Centerfor Biotechnology Information, which entry is incorporated herein byreference and refers to a G←→C transversion at the reference nucleotideposition, where the G-allele is the major allele and the C-allele is theminor allele). The present methods may detect a G-allele or a C-allelecorresponding to the polymorphism (i.e., a G-nucleotide or aC-nucleotide at the position associated with the rs518147). The presentmethods may detect whether a patient is homozygous or heterozygous for aG-allele or C-allele (i.e., whether the patient is GG, GC, or CC at thereference nucleotide position for rs518147).

The present methods may detect the polymorphism directly by analyzingchromosomal nucleic acid having the polymorphic variant sequence.Alternatively, the present method may detect the polymorphism indirectlyby detecting an isoform nucleic acid expressed from the polymorphicvariant sequence, by detecting an isoform polypeptide expressed from thepolymorphic variant sequence, or by analyzing the expression of anothernucleic acid or protein whose expression is regulated by the polymorphicsequence.

Examples

The following Example is illustrative and is not intended to limit theclaimed subject matter.

Example Association of Sertonin_(2c) Receptor Polymorphisms withAntipsychotic Drug Response in Schizophrenia

Abstract

The serotonin (5-HT)2C receptor (HTR2C) has been implicated inschizophrenia and response to antipsychotic drugs (APDs) through itsregulatory effect on dopamine release, interaction with scaffoldingproteins at the synapse, and other unknown mechanisms. Evidence fromgenetic association studies also implicates HTR2C in a variety ofneuropsychiatric diseases. We tested the association between HTR2Cpolymorphisms, Cys23Ser, −759C/T, and −697G/C, and treatment response in171 schizophrenic patients after treatment with APDs, mostly clozapine,for 6 months.

We confirmed that −759C/T, but not Cys23Ser was a cis-eQTL for HTR2Caccording to Braincloud data, an integrated database of genome-wide geneexpression and genetic control in human postmortem dorsolateralprefrontal cortex (DLPFC) of normal subjects. Ser23 was significantlyassociated with treatment response at 6 months (positive symptoms.X²=7.540, p=0.01; negative symptoms, X²=4.796, p=0.03) in male, but notin female patients. Haplotype analysis showed that −759C-Ser23maintained the same level of significant association with positivesymptom improvement (X²=6.648, p=0.01) but additive association withnegative symptom improvement (X²=6.702, p=0.01). Logistic regressionafter controlling forcovariates showed these haplotypic associationsremained significant with the same direction. Finally, a meta-analysiswas performed on six studies with accessible genotyping data for rs6318and treatment outcome. The overall odds ratio under fixed effect modelis 2.00 (95% CI, 1.38-2.91, p=0.0003) and under random effect model is1.94 (95% CI, 1.27-2.99, p=0.0024), In conclusion, HTR2C polymorphismswere associated with treatment response to clozapine in maleschizophrenic patients. HTR2C could be relevant to a broad range of thepsychopathology which responds to clozapine in schizophrenia.

Introduction

The (5-HT)2C receptor (HTR2C), located at Xq24, belongs to aseven-transmembrane-spanning G protein-coupled receptor superfamily andmediates phospholipase C activation and subsequent excitation of aphosphatidylinositol-calcium second messenger system in neurons [1-3].It is widely distributed in which are relevant to schizophrenia, such asthe ventral tegmentum, nucleus accumbens, striatum, prefrontal cortex,and hippocampus.

1. HTR2C Associated with Schizophrenia and Treatment Response Evidencefrom Neurobiology

HTR2C receptors have a tonic inhibitory effect on dorsal and ventralstriatal and conical dopamine (DA) release[4,5], modulate serotonergicactivity in the dorsal raphe[6], and suppress the increased serotoninand glutamate release in rat cortex following administration of the NMDAreceptor antagonist 3-(R)-2-carboxypiperazin-4 propyl-1-phosphonic acid(CPP)[7].

HTR2C receptors also have potent interactions with a variety ofscaffolding proteins, and play a key role in regulating synapsedevelopment and maintenance[8,9]. HTR2C interacts with severalPDZ-domain-containing proteins. These proteins are actively involved intargeting and trafficking transmembrane proteins, such as PSD95 (DLG4)MPDZ, and a tripartite complex, Veli3-CASK-Mint 1[9,10]. PSD95 formsmultimeric scaffold complexes at postsynaptic sites, enabling clusteringof receptors and associated signaling proteins, including the NMDAreceptor, a key component of the hypoglutamatergic deficit postulated inschizophrenia[11] and an essential contributor to memory[12] In additionto its role in scaffolding macromolecular glutamatergic signalingcomplexes. PSD-95 profoundly modulates metabotropic HTR2A and HTR2Cfunction. HTR2C is involved in the action ofcalcium/calmodulin-dependent serine protein kinases i.e. CASK, which isimportant for β-arrestin-dependent signaling[8]. Accumulated evidencesuggests a role of β-arrestin-mediated pathway in schizophrenia and themechanism of action of antipsychotic drugs (APDs) [8,13].

Some atypical APDs, e.g. clozapine, olanzapine, risperidone, andsertindole, are potent inverse agonists of both HTR2C and HTR2Areceptors, whereas, quetiapine, and aripiprazole are not [14,15]. On theother hand, some typical APDs, e.g. chlorpromazine, thioridazine,spiperone, and thiothixene, are HTR2C neutral antagonists. HTR2Cactivation and inhibition of dopamine (DA) efflux in the cortex andlimbic system indicate its involvement in cognition, psychosis, andaddictive behaviors[1,3,16,17]. Blockade of the constitutive activity ofHTR2C receptors has been implicated in enhancing cortical and limbic DArelease by some APDs [17].

There is indirect evidence for enhanced mesolimbic DA release inresponse to an amphetamine challenge in drug-nave schizophrenicpatients. This has been interpreted as evidence that schizophrenicpatients during an acute episode have increased presynaptic dopaminergicactivity[18,19]. If so, the ability of HTR2C agonists to reduce DArelease from terminals of VTA neurons in mesolimbic areas would beexpected to have an antipsychotic effect. This has been demonstrated invabicaserin[20]. In addition, reduced mRNA level of HTR2C has beenreported in postmortem prefrontal cortex tissues of schizophrenia[21]and bipolar disorder[22]. Thus, there is extensive evidence that HTR2Cis involved, in the neurobiology of schizophrenia and the action ofAPDs.

HTR2C Associated with Schizophrenia and Treatment Response: Evidencefrom Genetic Association Studies

Evidence from genetic association studies also implicates HTR2C in avariety of neuropsychiatric diseases. According to a comparative genuinestudy on DNA methylation from great ape evolution to recent humans, thefrequency of genetic variation is positively correlated with alterationsin promoter methylation[23]. The HTR2C has a well-characterized promoterregion harboring multiple polymorphisms suggesting their potentialimpact on CpG methylation and putative transcription factor binding,resulting in alteration of HTR2C expression. rs3813929 (−759C/T) andrs518147 (−697G/C) are the most widely-investigated HTR2C promoterpolymorphisms. −759C/T polymorphism is associated with antipsychoticinduced weight gain [24]. −759C/T or −697G/C, has also been linked totherapeutic response to APDs[25,26]. However the results arecontradictory with regard to gender and risk allele, rs6318, also knownas Cys23Ser, is a non-synonymous SNP which results in an amino acidsubstitution of cysteine to serine at position 23. This substitutioncould disrupt a disulfide bridge and potentially alter the structure orstability of the HTR2C protein. This functional polymorphism has beenfound to be associated with neuropsychiatric diseases such as anorexianervosa[27], unipolar and bipolar depression[28,29], and stress-relatedcortisol level [30]. It is unclear whether this SNP has any associationwith schizophrenia. A linkage study demonstrated no preferentialtransmission of either Cys or Ser alleles in 207 families withschizophrenia [31]. An association between the Cys23Ser and visualhallucinations and depression in schizophrenia patients is reported[32],but cannot be replicated by others[33,34]. Cys23Ser is also associatedwith chronic hospitalization[35], extrapyramidal side effects ofAPDs[36], psychotic symptoms in late-onset Alzheimer's disease[32], andmigraine with aura [37]. rs6318 has also been linked to alteredtreatment response to clozapine[38,39]. How Cys23Ser impacts on HTR2Cfunction is uncertain. The Ser23 variant has been associated withgreater constitutive activity, lower affinity, and alteredresensitization in some in vitro cell-line based functionalassays[40,41] but not others[42,43]. Since clozapine has high affinityfor HTR2C and acts as an inverse agonist[14], higher constitutiveactivity of Ser23 might mechanistically explain this association betweenrs6318 and treatment response to clozapine. The following studies onclozapine from other independent cohorts[44,45] [46] has failed toreplicate this association.

Due to the inconsistent relationship between HTR2C polymorphism andpsychopathology of schizophrenia and response to clozapine, theinconsistent results from functional activity assays, we examine allthree widely-investigated HTR2C SNPs as possible predictors of responseto treatment with APDs in schizophrenia in terms of positive and/ornegative symptoms. Male and female are analyzed separately in order todetermine whether the possible association has any gender preference.Finally a meta-analysis is performed to determine the overallassociation between HTR2C polymorphism and drug response (mainlyclozapine).

Materials and Methods

Subjects

The 171 (male/female, 115/56) patients with schizophrenia orschizoaffective disorder who participated in this study were part of anNIMH-sponsored extramural clinical research center at Case WesternReserve University School of Medicine and Vanderbilt University Schoolof Medicine. Details about recruitment and assessment of subjects havebeen reported previously [47]. Categorical treatment response wasevaluated at 6 week and 6 months, using the criteria based upon Kane etal[48]. A reduction of >20% in Brief Psychiatric Rating Scale (BPRS)total or subscale scores was considered as responder. In cases wherepatients were very close to the operational criteria for response (>15%or <25%), a reduction of at least one category on the Clinical GlobalImpressions (CGI) scale was considered in order to enhance thedefinition of response. In the present study, patients were treated withstandard doses of the following atypical antipsychotic drugs (clozapine,78%; melperone, 7.0%; risperidone, 3.8%; or olanzapine, 2.1%) or typicalantipsychotic drugs (9.0%). Antidepressants (14%) and mood stabilizers(5%) were used sparingly.

Genotyping

Blood samples for all the patients were processed with Qiagen genomicDNA extraction kit (Valencia, Calif., USA) at the Center for HumanGenetics Research Core at Vanderbilt University (Nashville, Tenn., USA).Taqman® assay for three SNPs, rs3813929 (−759C/T), rs518147(−697G/C),and rs6318 (Cys23Ser) in the HTR2C gene was performed on an ABI 7900HTSequence Detection System (Applied Biosystems, Foster City, Calif.,USA). Genotype data for rs498207 from some samples was provided by theClark Institute of Psychiatry (Toronto, Ontario, Canada). Call rates are95.32%, 98.83%, and 97.66% for −759C/T, −670G/C, and Cys23Serrespectively.

Mapping cis eQTL and Methylation QTL

BrainCloud allows the query of genome-wide gene expression data andtheir genetic control in human postmortem DLPFC of normal subjectsacross the lifespan[49]. We included all the SNP loci available within200 kb interval (100 kb up- and downstream) of HTR2C transcript. The pvalue corresponds to the regression coefficient based on the residualexpression level from the General Linear Model and the genotype obtainedfrom 140 postnatal Caucasian subjects. We also inquire a genome-wide DNAmethylation database, BrainCloud Methyl, derived from the sameBrainCloud subjects [50], to see if those identified cis eQTLs have anyimpact on DNA methylation and considered as methylation QTLs.

Statistical Analysis

We analyzed the males and females separately because of functionaluncertainty in heterozygous females due to either X chromosomeinactivation (XCI) or other confounding factors related to genderdifference, for example, a much stronger effect of the geneticassociation with clozapine-induced weight gain was observed in femalesubjects[51].

The relationship between genotypes and demographic variables wasanalyzed using chi-square (χ²) or ANOVA. P-values reported aretwo-tailed whenever applicable. Statistical significance was defined asp<0.05. As all results are considered exploratory, there was noadjustment for multiple comparisons. All individual SNP and haplotypeassociation testing was conducted with PLINK 1.0.7[52]. Otherstatistical analysis was done by SPSS. Haplotype association analysiswas performed for all possible combinations of SNPs (includingconsecutive and non-consecutive SNPs). For rs6318, genotypes werecollapsed into a dominant model, separated by Ser carrier ornon-carrier, Genotypes/haplotypes effects on the binary outcome wereanalyzed at 6 weeks, and 6 months by comparing, the frequencies ofalleles or genotypes between responder and non-responder groups usingthe following methods: Pearson's Chi-square, stepwise logisticregression controlled for multiple covariates including race, drug, ageof onset, baseline score of BPRS subcategories, and status ofearly-responder. ANCOVA was also performed to test the associationbetween HTR2C polymorphisms and percentage change (%) or absolute change(Δ) in BPRS total score and subcategories, after controlling for raceand the corresponding, baseline psychopathology. Linkage disequilibrium(LD) was visualized by Haploview. A SNP Annotation and Proxy Search(SNAP) was used to search proxy (tag) SNPs in LD with target SNPs fromHapmap database (release 21). Genotyping data from 1000Genome Databasewas extracted to confirm those tag SNPs are indeed in LD with targetSNPs. Meta-analysis was performed by R ‘meta’ package under R Studiointerface.

Results

Genotyping Quality Control and Demographic Data

Cys23Ser, −759C/T and −697G/C were genotyped for 171 Caucasian (118) andAfrican-American (53) patients with schizophrenia. (See Table 1A (MalePatients) and Table 1B (Female Patients)). The relationship betweengenotypes and demographic variables was analyzed using chi-square (χ²)or ANOVA. P-values reported are two tailed whenever applicable.Statistical significance was defined as p<0.05.

TABLE 1A (Male Patients) SNP ID rs3813929 rs518147 rs6318 P P Ser PGenotype T/T C/C value C/C G/G value Ser carrier non-carrier valueCounts 17 100 40 77 24 90 Frequency 0.14 0.86 0.34 0.66 0.22 0.78 TotalBPRS 27.88 ± 17.51 30.57 ± 12.08 0.43 32.3 ± 14.9 29.08 ± 11.78 0.234.92 ± 12.30 29.03 ± 12.89 0.04 Positive 4 items 9.41 ± 7.20 11.22 ±5.36  0.23 11.4 ± 5.74 10.73 ± 5.65  0.54 12.69 ± 4.14  10.59 ± 5.81 0.09 Positive 3 items 7.47 ± 5.91 9.04 ± 4.72 0.23 9.282 ± 4.91  8.55 ±4.94 0.45 10.25 ± 3.53  8.57 ± 5.04 0.13 Negative 3 items 3.94 ± 2.334.43 ± 3.01 0.53 4.15 ± 2.94 4.47 ± 2.91 0.58 4.65 ± 3.03 4.30 ± 2.870.59 Anxiety/Depression 4.69 ± 2.87 4.99 ± 3.67 0.75 5.39 ± 3.49 4.72 ±3.59 0.35 5.24 ± 3.70 5.00 ± 3.48 0.72 Age onset (year)   20 ± 6.2420.26 ± 5.12  0.86 20.75 ± 6.36  19.93 ± 4.60  0.43 22.78 ± 7.83  19.57± 4.27  0.01 Duration of illness (year) 11.71 ± 11.34 12.21 ± 7.45  0.8111.63 ± 9.50  12.41 ± 7.26  0.62 10.61 ± 8.63  12.42 ± 8.01  0.34 No. ofhospitalization 4.38 ± 4.24 6.66 ± 7.15 0.22 4.54 ± 4.09 7.28 ± 7.780.05 4.76 ± 4.13 6.56 ± 6.96 0.26 Treatment resistant (%) 52.9 70.0 0.1760 71.4 0.21 62.5 68.9 0.55 Baseline Unmedicated 81.3 76.9 78.4 77.10.88 72.7 78.0 0.6 (%) Clozapine treated (%) 82.4 74.0 78.7 85.0 72.70.34 83.3 74.4 0.28 Ethnicity¹ 0.004 0.19 0.04 Caucasian 12 67 24 55 1166 Afro-American 5 33 16 22 13 24 ¹Self-described ethnicity.

TABLE 1B (Female Patients) SNP ID rs3813929 rs518147 rs6318 P P Ser non-P Genotype T/T C/C value C/C C/G G/G value Ser carrier carrier valueCounts 3 41 7 24 21 20 33 Frequency 0.07 0.93 0.13 0.46 0.4 0.38 0.62Total 28.33 ± 9.29  34.27 ± 11.48 0.39 30.86 ± 11.85 35.71 ± 11.75 33.81± 10.61 0.8  34.9 ± 14.41 33.82 ± 8.40  0.73 BPRS Positive 4 8.33 ± 6.1112.66 ± 4.52  0.12 11.14 ± 6.67  13.58 ± 4.07    12 ± 4.89 0.91 12.9 ±5.18 12.15 ± 4.76  0.58 items Positive 3   7 ± 5.29 11.03 ± 3.93  0.1  10 ± 6.83 11.78 ± 3.57  10.43 ± 4.06  0.83 11.2 ± 4.95 10.5 ± 4.2 0.58 items Negative 3 3.33 ± 3.22 4.39 ± 3.31 0.6   4 ± 3.74  4.5 ± 2.704.76 ± 3.90 0.61 4.43 ± 3.01 4.62 ± 3.46 0.84 items Anxiety/ 7.33 ± 4.736.42 ± 4.12 0.71 6.14 ± 3.53 5.75 ± 3.92 6.91 ± 4.44 0.48 6.05 ± 3.946.27 ± 4.03 0.85 Depres- sion Age onset 20.33 ± 4.04  21.55 ± 5.85  0.73  21 ± 4.08 21.30 ± 5.47  21.29 ± 5.68  0.99 21.79 ± 4.85  21.58 ± 6.27 0.9 (year) Duration   14 ± 15.59 11.63 ± 7.27  0.62 15.29 ± 11.41 14.74± 7.81    10 ± 6.51 0.1 13.05 ± 6.96  12.49 ± 8.73  0.81 of illness(year) No. of   3 ± 2.83 8.11 ± 8.74 0.42  4.5 ± 3.14   7 ± 5.51  9.4 ±10.21 0.35 7.94 ± 9.50 7.03 ± 6.79 0.71 hospitali- zation Treatment 66.770.7 0.88 71.4 79.2 61.9 0.44 80 69.7 0.41 resistant (%) Baseline 100.063.6 0.29 66.7 78.9 61.1 0.49 60.0 72.4 0.4 Unmedi- cated (%) Clozapine66.7 85.4 0.1 71.4 87.5 90.5 0.003 90.9 75.0 0.14 treated (%) Ethnicity¹0.2 0.62 0.08 Caucasian 3 26 6 16 15 12 27 Afro- 0 15 1 8 6 8 6 American¹Self-described ethnicity.

Hardy-Weinberg equilibrium (HWE) test for female Caucasian subjects wasnot significant (p<0.01), except for rs3813929 (p=0.003), due to a lackof patients with the heterozygous genotype. We further genotyped 300more schizophrenic subjects and HWE test wasn't significant (p=0.45),suggesting this departure from HWE is related to small sample size(n=53) and low minor allele frequency (MAF) for rs3813929 (14.93% in1000Genome data; 10.35% in our samples) in the female Caucasians. (SeeTable 2).

TABLE 2 Summary of genotypic distribution of three mostly investigatedHTR2C SNPs in Caucasians from 1000Genome, Braincloud, and our data.rs3813929 Female Male Data source TT TC CC T C tag SNP 1000 genome 2(1%)   56 (27.9%) 143 (71.1%)  32 (18%)   146 (82%)   rs3813929Braincloud 1 (3.1%)  5 (15.6%) 26 (81.3%) 17 (21.8%) 61 (78.2%)rs12846241 Our sample  3 (10.3%) 0 (0%)   26 (89.7%) 12 (15.2%) 67(84.8%) rs3813929 rs518147 Female Male Data source CC CG GG C G tag SNP1000 genome 16 (8%)   92 (45.8%) 93 (46.3%) 67 (37.6%) 111 (62.4%) rs498207 1000 genome 17 (8.4%)  91 (45.3%) 93 (46.3%) 66 (37.1%) 112(62.9%)  rs518147 Braincloud 3 (9.4%) 12 (37.5%) 17 (53.1%) 39 (50%)  39 (50%)   rs498207 Our sample 3 (12%)  8 (32%)  14 (56%)   23 (33.8%)45 (66.2%) rs498207 Our sample  6 (16.2%) 16 (43.2%) 15 (40.6%) 24(30.4%) 55 (69.6%) rs518147 rs6318 Female Male Data source CC CG GG C Gtag SNP 1000 genome 4 (2%)   51 (25.4%) 146 (72.6%)  32 (18%)   146(82%)   rs6318 Braincloud 2 (6.7%)  4 (13.3%) 24 (80%)   19 (24.4%) 59(75.6%) rs5987834 Our sample 2 (4.8%) 12 (28.5%) 28 (66.7%) 14 (16.7%)70 (83.3%) rs6318 Three datasets were genotyped by illuminanext-generation sequencing, illumina Beadarray, and ABI Taqman assay,respectively. Data was presented as genotype count (% in Female or Malesubjects).

The haplotype view was generated for our sample and 1000Genome sampleseparated by ethnicity. (See FIG. 3). The LD patterns (r²) arecomparable and consistent between the two samples, suggesting ourgenotype data are reliable and representative. Interestingly, thegenotype frequency between rs3813929 and rs6318 are very similar acrossthree Caucasian datasets (1000Genome, Braincloud, and ours) according toTable 2. Although the D′ was equal to one (data not show), the r² wasvery low. (See FIG. 3), suggesting gene conversion happened. We indeed,observed a recombination event, >40 cM/Mb recombination rate, in theboundary between promoter and transcriptional starting, based onregional LD plot generated from SNAP (data not shown).

In the male group, the age at onset for the Ser23 carriers wassignificantly older than that for non-carriers (p=0.009). (See Table1A). This difference was not observed in the female group (p=0.899).(See Table 1B). There was no significant difference in the proportionsof patients who were treatment resistant or unmedicated at baselinebetween the genotypes for each SNP. Duration of illness and number ofprevious hospitalization also did not differ. Although Ser carriers hada higher total BRPS score in the male patients (borderline significance,p=0.04), there was no significant difference with regard to thesubcategories of psychopathology including positive, negative, andanxiety/depression subscales. Race, drug, age of onset were included ascovariates in the following regression analysis.

Rs3813929 is cis-eQTL for HTR2C

We successfully identified the tag SNP, which are in LD with thecandidate SNPs (r²>0.85) by SNAP, based on earlier Hapmap data (21) inCaucasians. We confirmed those tag SNP are indeed, in LD with threecandidates by a haploview analysis of the acquired genotype data from1000Genome. (See FIG. 1). rs12846241 (r²=0.96), rs498207 (r²=0.98) andrs5987834 (r²=0.86) are the tag SNPs, genotyped in Braincloud, forrs3813929, rs518147 and rs6318, respectively, in Caucasians. A singleexpression probe selected for HTR2C is located in the 3′ end of mRNA.(See FIG. 1, which represents the major form of HTR2C cloned frommultiple brain regions according to NCBI Aceview). For the cisassociation analysis, we initially identified six SNPs associated withgene expression (p<0.05), (See FIG. 1). Examining postnatal subjectsseparated by gender, this association becomes stronger in males, but notin females and more cis eQTLs were identified. (See Table 3).

TABLE 3 A list of potential cis-eQTLs and methylation-QTLs identified byBraincloud and Brainmethyl database. Minor Observed SNP in Cis-eQTLMethylation-QTL SNP ID Allele Genotype Location promoter MAF (M/F) TagSNP (AA/EUR) (AA/EUR) rs489736 A A/G Intergenic-5′ No 0.382/0.3160.058/0.045 0.002/0.002 rs11798015 G G/A Intergenic-5′ No 0.152/0.154rs3813929 (LD)   NA/0.004   NA/0.000 rs547617 G G/A Intergenic-5′ No0.371/0.313 rs518147 (LD) 0.162/0.043 0.003/0.021 rs3795182 G G/Anear-gene-5′ Yes 0.185/0.149 NA NA rs521018 G G/T near-gene-5′ Yes0.376/0.309 NA NA rs498207 G G/A near-gene-5′ Yes 0.376/0.309 rs518147(LD) 0.267/0.043 0.014/0.096 rs3813928 A A/G near-gene-5′ Yes0.180/0.149 rs3813929 (LD) NA NA rs3813929 T T/C near-gene-5′ Yes0.180/0.149 −759 (C/T) NA NA rs518147 C C/G untranslated-5′ Yes0.371/0.311 −697 (G/C) NA NA rs12846241 G G/T intron No 0.180/0.152rs3813929 (LD) 0.116/0.006 0.205/0.009 rs12690355 G G/A intron No0.191/0.154 rs3813929 (LD) NA/0.016   NA/0.028 rs6318 C C/G exon No0.180/0.147 cys23ser NA NA rs4911871 G G/A intron No 0.202/0.179  NA/0.009   NA/0.002 rs6644093 T T/G intron No 0.146/0.132   NA/0.004  NA/0.007 rs5987834 T T/C Intergenic-3′ No 0.185/0.142 rs6318 (LD)0.914/0.601 0.561/0.944 MAF = minor allele frequency. M = male; F =female. AA = African American; Eur = European. NA represents data notavailable or not reported due to low MAF resulting in unreliable data.r² > 0.85 in LD. SNPs are located in promoter region and putative CpGIsland. Rs6318 and its tag SNP, rs5987834, exclusively available inBraincloud were negative controls. p < 0.01 is cut-off value forcis-eQTLs. 0.01 > P < 0.05 suggest a possible impact of that SNP on geneexpression.

This discrepancy could be a result of low MAF in the Braincloud femalesample, XCI, or other confounding factors related to gender difference.All the identified cis eQTLs are listed in Table 3. Most of them areaggregated at the promoter or 5′UTR regions as expected. (See FIG. 1).rs3813929, tagged by rs12846241, has been confirmed as cis-eQTL, forHTR2C. The T genotype has a lower mRNA level than C genotype (p=0.006),which is in conflict to some reported in vitro gene-reporterassays[53,54]. rs518147, tagged by rs498207, also suggests potentialimpact on gene expression (p=0.043), but not as strong as rs3813929.This finding echoed the earlier EMSA finding [55] that rs3813929, butnot rs518147, altered DNA-protein interactions, leading to change inexpression of HTR2C. Haplotype analysis of rs3813929-rs518147, tagged byrs12846241-rs498207, showed that male Caucasians with C-G (n=39) and C-C(n=22) genotypes had a significantly higher expression of HTR2C thanthose with T-C (n=17) genotype (corresponding p=0.007 and p=0.050).Since rs3813929 and rs518147 are located in a putative CpG island. (SeeFIG. 1), we also evaluated the relationship between the geneticpolymorphism and % methylation in this CpG site, in BrainCloud Methyl.We confirmed this significant association between rs3813929 genotype andDNA methylation (p=0.0005 for rs11798015 or 0.009 for rs12846241).Whereas for rs518147, this association has a borderline significance(p=0.096 by rs498207 or 0.021 by rs547617). rs6318, tagged by rs5987834,has no impact on gene expression and % so methylation. In rs3813929, Tbut not C, allele was associated with lower expression and highermethylation rate. We emphasize this association between rs3817929 andDNA methylation because the epigenetic markers may not be reproduced inin vitro gene-reporter system which is commonly used to confirm thefunctional activity of a putative genetic variant in a promoter region.The same trend of association, though not always significant due tolimited number of subjects, was observed, in Braincloud African-Americansample (see Table 3), suggesting that rs3813929 is a functional variantfor HTR2C independent of ethnicity. It is noted that all the abovefindings do not apply for Braincloud females.

Genotype/Haplotype Association Study

Based on the previous studies (genetic and functional) and our cis eQTLfindings, rs3813929, rs6318, rs518147, and the combinations of two orall three, were targets for the subsequent genotype-phenotypeassociation study. Since in vitro functional assays indicated thatrs3813929 and rs6318 have a significant impact on HTR2C activity throughdistinct mechanisms, we further explored if −759C-Ser, “a supercombination”, as we proposed to produce a greater expression of theconstitutively more active form of HTR2C, may demonstrate an evenstronger association with dichotomous symptom improvement in an additivemode, after treatment with the HTR2C inverse agonists or antagonistsstudied here.

A significant association between rs6318 and dichotomous treatmentresponse was observed only in the male group for both positive symptoms,X²=7.540, p=0.006; and negative symptoms, X²=4.796, p=0.029. (See Table4).

TABLE 4 Haplotype association analysis of HTR2C SNPs with treatmentresponse by Chi-square test in male patients. Male Only BPRS Positive 4Items BPRS Positive 3 Items Frequency in Frequency in HaplotypeResponder/non- Responder/non- SNP ID Haplotype frequency responder X²/Presponder X²/P rs3813929 (−759) C 0.861 0.810/0.677 0.881/0.3630.851/0.851 0/1 rs518147 (−697) C 0.336 0.452/0.263  3.84/0.0500.447/0.255 3.782/0.052 rs6318 (Cys23Ser) Ser carrier 0.22 0.289/0.1612.406/0.121  0.34/0.106  7.54/0.006 rs3813929-rs518147 C-C 0.1970.262/0.140 2.304/0.129 0.298/0.106 5.343/0.021 rs518147-rs6318 C-Ser0.176 0.244/0.125 2.315/0.128 0.283/0.085  6.07/0.014 rs3813929-rs6318C-Ser 0.202 0.268/0.161 1.674/0.196 0.326/0.106 6.648/0.010rs3813929-rs518147-rs6318 C-C Ser 0.171 0.238/0.123  2.26/0.1330.277/0.085 5.817/0.016 BPRS Negative 3 Items Anxiety/Depression 3 ItemsFrequency in Frequency in Responder/non- Responder/non- SNP ID responderX²/P responder X²/P rs3813929 (−759) 0.838/0.816 0.059/0.807 0.64/0.8480.011/0.916 rs518147 (−697) 0.405/0.291 1.298/0.255 0.34/0.3480.007/0.936 rs6318 (Cys23Ser) 0.308/0.125 4.796/0.029 0.25/0.1960.414/0.520 rs3813929-rs518147 0.243/0.109 2.917/0.088 0.18/0.1960.039/0.844 rs518147-rs6318 0.243/0.093 3.828/0.050 0.184/0.178 0.006/0.941 rs3813929-rs6318 0.306/0.093 6.702/0.010 0.225/0.2  0.084/0.772 rs3813929-rs518147-rs6318 0.222/0.091 3.064/0.080 0.18/0.1740.006/0.938 Positive 4-item includes suspiciousness, hallucinatorybehavior, unusual thought content, and conceptual disorganization.Positive 3-item = Positive 4-item without “concept disorganization”.Male and female subjects were analyzed separately.

Haplotype analysis showed that −759C-Ser maintained the same level ofsignificant association with positive symptom improvement (X²=6.648,p=0.010) and additive association with negative symptom improvement(X²=6.702, p=0.010) when comparing to the association between Ser aloneand dichotomous variables. (See Table 4). Logistic regression aftercontrolling for race, age of onset, and drugs showed the haplotyperesults remained similar significance in the male group. None of thecovariates had a significant impact on this association (p>0.05). Sincebaseline BPRS level may be a strong predictor of treatment response, weincluded it as a covariate. Ser alone still showed the most significantassociation with positive symptom improvement across all the haplotypecombinations (OR=3.63; p=0.030). (See Table 5).

TABLE 5 Haplotype association of HTR2C SNPs with treatment responseanalyzed by logistic regression. Data were presented as Odds Ratio/Pvalue. Male Only BPRS Positive 3 Items BPRS Negative 3 Items Add CoverAdd Cover (Early Add Cover (Early Add Cover Haplotype response (Baselineresponse (Baseline SNP ID Haplotype Frequency status) BPRS) status)BPRS) rs3813929 (−759) C 0.861 1.25/0.724 1.02/0.979 1.81/0.4131.09/0.891 rs518147 (−697) C 0.336 1.85/0.205 2.05/0.134 1.63/0.3711.75/0.25  rs6318 (Cys23Ser) Ser carrier 0.22  3.7/0.034 3.63/0.0303.69/0.048 2.99/0.057 rs3813929-rs518147 C-C 0.197 3.05/0.074 2.83/0.0813.83/0.054 2.69/0.113 rs518147-rs6318 C-Ser 0.176 3.51/0.057 3.33/0.061 4.6/0.036 3.19/0.075 rs3813929-rs6318 C-Ser 0.202  3.6/0.040  3.4/0.0424.89/0.025 3.94/0.030 rs3813929-rs518147-rs6318 C-C-Ser 0.171 3.44/0.0623.21/0.067 4.52/0.040 2.97/0.100 No. of subjects (EUR/AA) 72/35  69/24 74/33  71/26  Positive 3-item includes suspieiousness, hallucinatorybehavior, and unusual thought content. Male and female subjects wereanalyzed separately. 5A-B, Data was adjusted for Race and Drug. 5C-D,Data was adjusted for baseline score for psychopathology or status ofearly responder at 6-week assessment.

−759C-Ser showed the strongest relationship to negative symptomimprovement (OR=3.94; p=0.030). The low MAF (0.09) of −759C/T inAfrican-Americans and relatively lower call rates (95.32%), as comparedto other SNPs (98.83% and 97.66%) in our study, may account for notobserving the additive association between −759C-Ser and positivesymptom improvement. rs518147 had no add-on effect on this associationwhen comparing rs3813929-rs518147-rs6318 with rs3813929-rs6318 orcomparing, rs6318-rs518147 with rs6318 alone. However,rs3813929-rs518147 demonstrated a trend of association with dichotomousvariables (corresponding OR=2.83, p=0.081 or OR=2.69, p=0.1 for positiveor negative symptoms) despite rs518147 or rs3813929 alone showing nosignificant association at all. (See Table 5), suggesting that rs518147does have a minor functional impact on HTR2C biology. This positivefinding with dichotomous positive 3-item improvement is not found whenusing dichotomous positive 4-item improvement which includes conceptualdisorganization. 11% of patients were not available for the 6-monthclinical assessment. The majority of the drop-outs wereAfrican-Americans (78.9%). The drop-outs may potentially underestimatethe true association between the genetic variants and treatmentresponse. Although this drop-out may be unrelated to response status at6-week assessment (50% drop-offs are early responders), we stillconsidered it as a covariate in our stud. Once again. Logisticregression after adding this covariate showed a similar result with thesame trend. (See Table 5). This pattern of haplotype association was notobserved in the 6-week assessment. All of the above significant findingswere only observed in the male patients except for a borderlinesignificance for rs6318 associated with negative symptom improvement infemale (X²=3.9, p=0.048). (See Table 6).

TABLE 6 Haplotype association analysis of HTR2C SNPs with treatmentresponse by Chi-square test in female patients and all patients. BPRSPositive 4 Items BPRS Positive 3 Items BPRS Negative 3 Items Frequencyin Frequency in Frequency in Haplotype Responder/non- Responder/non-Responder/non- SNP ID Haplotype frequency responder X²/P responder X²/Presponder X²/P Female Only rs3813929 (−759) C 0.936  0.95/0.8462.056/0.152  0.95/0.833 2.403/0.121 0.833/0.941 1.764/0.184 rs518147(−697) C 0.364 0.333/0.438 0.689/0.346 0.326/0.438 1.003/0.3170.385/0.370 0.0161/0.899  rs6318 (Cys23Ser) Ser carrier 0.3970.296/0.438 1.764/0.164  0.32/0.375 0.263/0.608 0.4687/0.25   3.9/0.048rs3813929-rs518147 C-C 0.25 0.211/0.231 0.037/0.847 0.211/0.2080.000/0.984 0.136/0.235 0.826/0.363 rs518147-rs6318 C-Ser 0.2130.188/0.219 0.117/0.732 0.196/0.188 0.008/1    0.192/0.174 0.038/0.846rs3813929-rs6318 C-Ser 0.413  0.3/0.462 1.777/0.183  0.3/0.4170.905/0.341 0.417/0.284 0.935/0.333 rs3813929-rs518147-rs6318 C-C-Ser0.205 0.18/0.219 0.187/0.686 0.188/0.186 0/1 0.179/0.174 0.003/0.959Male + Female rs3813929 (−759) C 0.894 0.878/0.668 0.0416/0.839 0.8970.845 0.937/0.333 0.836/0.865 0.245/0.621 rs518147 (−697) C 0.3490.388/0.326 0.774/0.379 0.367/0.329 0.623/0.43  0.397/0.327 0.634/0.361rs6318 (Cys23Ser) Ser carrier 0.305 0.293/0.261 0.231/0.631  0.33/0.2152.89/0.089 0.377/0.183 8.122/0.004 rs3813929-rs518147 C-C 0.220.236/0.169 1.195/0.274 0.259/0.141 3.302/0.068 0.203/0.157 0.520/0.471rs518147-rs6318 C-Ser 0.194 0.214/0.159 0.863/0.353 0.239/0.1273.539/0.060 0.222/0.13  2.378/0.123 rs3813929-rs6318 C-Ser 0.2940.284/0.256 0.160/0.689 0.314/0.211 2.093/0.148 0.35/0.171  6.248/0.0124rs3813929-rs518147-rs6318 C-C-Ser 0.187 0.206/0.157 0.737/0.3910.231/0.126 3.181/0.075 0.203/0.128 1.629/0.202

Since we have a relatively lower case number and unphased genotype datafor females, it is not possible to reach a conclusion for individual SNPassociation and haplotype association in females. Subsequent ANCOVA teston Δ change or % change (data not shown) in symptom improvement aftercontrolling for race, drugs, and the corresponding baselinepsychopathology indicated that Ser carriers had a significantimprovement in positive and negative symptoms (p=0.025 and 0.019,respectively) after 6 month treatment in the male patients. (See Table7).

TABLE 7 ANCOVA test on the association between HTR2C SNPs with absolutechange (Δ) in symptom improvement with gene polymorphism as theindependent variable and race and corresponding baseline psychopathologyas the covariates in a general linear model. Male only SNP ID rs3813929rs518147 Genotype T/T C/C F/P^(&) C/C C/G Counts 17 95 39 NA Frequency0.140 0.860 0.342 NA Total_6 Mon  −8.47 ± 13.25  −7.06 ± 12.08 0.748/0.389^(#) −10.36 ± 11.62 NA Total_6 Wk −7.00 ± 8.23  −5.63 ±11.92 3.93/0.05^(# )  −8.25 ± 11.13 Positive 4 items_6 Mon −2.06 ± 4.66−2.65 ± 5.76  0.01/0.914^(#) −3.56 ± 4.77 NA Positive 4 items_6 Wk −2.76± 4.66 −2.03 ± 5.27 1.99/0.161 −2.83 ± 4.60 Positive 3 items_6 Mon −1.35± 4.36 −2.25 ± 4.77 0.04/0.835 −2.97 ± 4.33 NA Positive 3 items_6 Wk−2.24 ± 4.31 −1.61 ± 4.23 1.81/0.181 −2.33 ± 4.10 Negative 3 items_6 Mon−0.41 ± 3.87 −0.85 ± 2.75 0.15/0.700 −1.25 ± 3.62 NA Negative 3 items_6Wk  0.00 ± 2.78 −0.15 ± 2.84 0.14/0.708 −0.03 ± 2.36 SNP ID rs518147rs6318 Genotype G/G F/P Cys/Ser + Ser/Ser Cys/Cys F/P Counts 73 24 89Frequency 0.658 0.217 0.783 Total_6 Mon  −5.64 ± 12.30 1.30/0.258 −12.65± 9.09   −6.16 ± 12.60  2.82/0.096  Total_6 Wk  −5.39 ± 12.57 0.67/0.413 −9.58 ± 11.63  −6.20 ± 12.12 0.08/0.784 Positive 4 items_6 Mon −2.01 ±5.92 0.99/0.322 −5.22 ± 5.04 −2.09 ± 5.66 3.66/0.025 Positive 4 items_6Wk −1.77 ± 5.45 0.92/0.338 −3.19 ± 4.52 −2.03 ± 5.39 0.08/0.772 Positive3 items_6 Mon −1.61 ± 4.86 0.62/0.367 −4.74 ± 4.26 −1.59 ± 4.775.19/0.025 Positive 3 items_6 Wk −1.36 ± 4.28 1.08/0.302 −2.62 ± 3.97−1.59 ± 4.37 0.14/0.705 Negative 3 items_6 Mon −0.52 ± 2.51 2.64/0.107−2.04 ± 2.79 −0.52 ± 2.99 5.73/0.019 Negative 3 items_6 Wk −0.18 ± 3.050.05/0.819 −0.54 ± 2.21 −0.14 ± 3.06 0.67/0.416 Female only SNP IDrs3813929 rs518147 Genotype T/T C/C F/P C/C C/G Counts 3 40 7 23Frequency 0.068 0.932 0.135 0.462 Total_6 Mon −7.33 ± 8.33   −9.81 ±10.82 0.29/0.595  −9.71 ± 14.10  −9.88 ± 10.53 Total_6 Wk −3.33 ± 3.22  6.68 ± 12.19  0.37/0.0.549^(#) −9.43 ± 7.85  −8.32 ± 14.05 Positive 4items_6 Mon 0.33 ± 6.11 −3.75 ± 4.10 1.90/0.178 −2.43 ± 5.41 −3.71 ±3.62 Positive 4 items_6 Wk 0.33 ± 5.77 −2.55 ± 4.44 0.92/0.344 −3.71 ±5.74 −2.56 ± 4.74 Positive 3 items_6 Mon 0.33 ± 4.51 −3.42 ± 3.642.00/0.168 −2.14 ± 5.34 −3.00 ± 3.37 Positive 3 items_6 Wk 1.00 ± 5.29−2.22 ± 4.04 1.41/0.242 −3.00 ± 5.69 −1.78 ± 4.58 Negative 3 items_6 Mon−1.67 ± 3.06  −0.59 ± 2.45 0.73/0.400  1.57 ± 5.53 −0.53 ± 2.40 Negative3 items_6 Wk 1.33 ± 2.31 −0.93 ± 2.62 2.03/0.162  0.14 ± 2.91 −1.00 ±2.68 SNP ID rs518147 rs6318 Genotype G/G F/P Cys/Ser + Ser/Ser Cys/CysF/P Counts 21 20 34 Frequency 0.404 0.382 0.816 Total_6 Mon −11.17 ±11.15 0.22/0.802 −10.31 ± 13.28 −11.45 ± 9.93  0.06/0.811 Total_6 Wk −7.76 ± 11.84 0.24/0.786  −9.73 ± 12.97  −8.00 ± 12.20 0.22/0.641Positive 4 items_6 Mon −4.33 ± 4.83 0.74/0.483 −3.31 ± 3.61 −4.10 ± 4.64 0.44/0.512^(#) Positive 4 items_6 Wk −2.76 ± 4.00 0.70/0.502 −3.41 ±4.35 −2.56 ± 4.47 0.45/0.506 Positive 3 items_6 Mon −4.06 ± 4.151.19/0.315 −2.53 ± 3.74 −3.59 ± 4.06  1.06/0.311^(#) Positive 3 items_6Wk −2.71 ± 3.24 1.03/0.366 −2.55 ± 4.19 −2.24 ± 4.01 0.11/0.739 Negative3 items_6 Mon −0.51 ± 3.15 0.81/0.454 −1.75 ± 4.09 −0.38 ± 2.902.46/0.124 Negative 3 items_6 Wk −1.14 ± 2.83 0.49/0.817 −1.45 ± 2.88−0.62 ± 2.78 2.26/0.139 Clinical assessment was performed at 6 week and6 month. Data was presented as Mean ± SD of Δ change from baselinescore. ^(&)represents F statistic and p value calculated from ANCOVA;^(#)represents the result from Levene's test of equality of errorvariances with p < 0.05, which against the null hypothesis that theerror variance of the dependent variable is equal across groups,suggesting ANCOVA assuming homogeneity of variance is rejected.

These analyses suggest that HTR2C could be relevant to response to APDsfor many types of psychopathology. Neither −759C/T nor −697G/C alonewere significantly associated with symptom improvement.

Meta-Analysis

In order to review and elucidate the general relationship between HTR2Cpolymorphisms and drug response to clozapine, a Pubmed search wasconducted using Medline databases from 1966 to February, 2014. Theprocedure for meta-analysis is detailed in the Supplemental Informationpresented below. Six studies from Table 8, including ours, withaccessible genotyping data for rs6318 and binary outcome for symptomimprovement, were included.

TABLE 8 Previous association studies of HTR2C polymorphisms withtreatment response to antipsychotics, mainly clozapine, inSchizophrenia. No. of Subjects Study Responder/Non- Reference(Male/Female) Antipsychotics Ethnicity duration Genetic VariantsResponder Sodhi MS* 162 (unclear) Clozapine only Caucasian 3 monthsrs6318 103/59 (1995) Masellis M* 185 (132/53) Clozapine only Caucasian/6 months rs6318 72/67 for (1998) African- Caucasian: 20/19 American forAfrian Americans Arranz MJ 200 (unclear) Clozapine only Caucasian 3months −330(GT)/−244(CT); 133/67 (2000) rs6318 Reynolds 117 (58/59)Chlorpromazine Chinese 2.5 months   rs3813929  86/90 GP (2005) (56.4%);risperidone (Han) (36.8%); clozapine (3.4%); fluphenazine (2.6%);sulpiride (1%) Ikeda M 120 (58/62) Risperidone only Japanese 2.5months   rs3813929; not available (2008) rs518147 Need AC 524 (CATIEolanzapine, Caucasian/ Variable 30 tag not available (2009) Phase 1)perphenazine, African- (up to 18 SNPs in quetiapine, risperidoneAmerican months) HTR2C and ziprasidone Liu B-C 130 (45/85) Risperidoneonly Chinese 2 months rs3813929; not available (2010) (Han) rs518147;rs1023574; rs9698290; rs6318 Vehof J* 329 (260/69); Clozapine (9.1%);Caucasian variable rs3813929; 247/82 (2012) actual number is Olanzapine(24.3%); rs6318 293 (—/—) for Risperidone (22.8%); rs3813929 andQuetiapine (5.5%); 297 for rs6318 Haloperidol (7.3%); Multiple (11.2%);Aripiprazole (1.5%); others (6.7%) Malhotra 66 (49/17) Clozapine onlyCaucasian 2.5 months   rs6318  18/48 AK* (1996) Rietschel 152 (76/76)Clozapine only Caucasian variable rs6318 110/42 M* (1997) (Avg = 2.0months) Li & 171 (115/56) Clozapine (78%); Caucasian/ 6 weeks &rs3813929; 75/63 (positive Meltzer* Olanzapine (2.1%); African- 6 monthsrs518147; symptom at 6 (2014) Risperidone (3.8%); American rs6318 month)Melperone (7.0%); Others (9.0%) *represents studies included in themeta-analysis

Due to insignificant heterogeneity between studies (Cochran's Q=6.03,p=0.30; I²=0.17 (95% CI, 0.00 to 0.62), we report the overall odds ratiowith the fixed effect model is 2.00 (95% CI, 1.38-2.91, p=0.0003) andthat with the random effect model is 1.94 (95% CI, 1.27-2.99, p=0.0024).(See FIG. 2). QUANTO 1.2 was used to calculate the power of the test.The mode of inheritance for rs6318 was dominant, Kp=0.30 (populationrisk) since the prevalence of treatment responders to clozapine in theschizophrenia population is ˜30%. The Ser carriers were found to have afrequency of 0.15 to 0.45 according to Table 9.

TABLE 9 Allele frequency of mainly validated HTR2C SNPs from East Asian(ASN), European (EUR), and African (AFK) population in 1000 Genomeproject. EUR ASN (CEU, TSI, FIN, AFK (YRI, Gender SNP ID (CHB, JPT,CHS)GBR, IBS) LWK, ASW) Male rs3813929 16.7% 18.0% 3.5% rs518147 18.1% 37.1%43.5% rs6318 1.4% 18.0% 47.0% Female rs3813929 13.7% 14.9% 1.9% rs51814715.1% 31.1% 34.0% rs6318 1.1% 14.7% 37.8% Allele frequency of mainlyvalidated HTR2C SNPs from East Asian (ASN), European (EUR), and African(AFK) population in 1000Genome project. Original genotype data areretrieved from 1000 Genome database at its website.

If Ser carriers increases the odds of having treatment response by 2.0,and 216 responders and 738 non responders were genotyped, the power(chance) to detect an association with significance p<0.01 was over 90%.

Discussion

We summarize the published association studies between HTR2C geneticvariants and treatment response for APDs in schizophrenic patients inTable 2. There is no consistent result across all the studies; however,some potential replicable finding can be extracted. For example, thefollowing, replication studies[44,56] to Sodhi′[38] show no statisticalsignificance, but the individual p value is close to 0.05 and doesreveal a trend for association (Masellis, p=0.18 and Vehof, p=0.13). Ourmeta-analysis on six original studies[38,44-46,56] suggests that HTR2C.Cys23Ser is associated with symptom improvement after treatment withclozapine. This is an extension of a previously reported meta-analysison this topic[57]. Studies of a single APD (or of one dominant) are morelikely to reach a positive finding than studies based on diversetreatments. APDs may have distinct effect on HTR2C, and other receptors,leading to differential degree of symptom improvement. However, ourfindings could also mean that the results reported here do generalize,at least, to the drugs studies here or the entire class ofserotonin-dopamine receptor antagonists, of which clozapine is theprototype. One of its strengths in this study is that the majority ofthe patients were unmedicated at the time of assessment, providing dataon the basal level of psychopathology not affected by drug treatment.Only the perspective treatment, mainly association with the clozapinewas responsible for the symptom improvement.

A likely explanation for the inconsistent results among pharmacogeneticstudies of clozapine response is the heterogeneity in definition andcharacterization of phenotype or endophenotype, the magnitude ofimprovement evaluated with different scale system including PANSS[58],BPRS[59], CGI-1[60], et al, some with relative small range of responseoutcomes, frequency of genetic variants in distinct, ethnical groups,different subtypes of APDs which may or may not related to HTR2Cpharmacology, the time of assessment for treatment response based onpersonal clinical experience, and statistical methods (dominant,recessive or others, chi-square or regression model with covariates).Patients who do not respond well are less likely to continue anantipsychotic and will eventually drop out or switch to another APD.This would Favor negative results.

Another possible explanation for discrepant findings is that HTR2C mayexert different or even opposite activities in the brain depending onother genetic factor[61], which are relevant to the specificneuropsychiatric disease or specific phenotype/endophenotype. Numerousstudies using GWAS or candidate gene suggest the genetic mutations frommultiple genes may account for the association with the trait.Therefore, to analyze multiple functional polymorphisms together,instead of individually, may lead to a better understanding of thegenetic burden on the phenotype and avoid discrepant findings inassociation studies of individual SNP association. Despite −759C-Ser mayshow some additive effect on this backbone (Cys23Ser) association, thisstudy, including the result of the meta-analysis, strongly indicatedthat rs6318, but not promoter polymorphisms, is the major geneticcontributor in modulating symptom improvement to clozapine. However forAPI) induced weight gain, the consistent finding is −759C/T associatedwith APD-induced weight gain[24,61].

The drug target for symptom improvement (or treatment response) inschizophrenia mainly involved Cys23Ser. Some in vitro functional studiesmay partially give an explanation. Okada[40] reported functionaldifferences between the two alleles of rs6318 by comparing theirreceptor-binding profiles and ability to mobilize calcium in COS-7 cellsand by comparing their constitutive activities using a novel procedurefor in sin, reconstitution. Ser23 receptor displayed constitutively moreactivity than Cys23. Thus, it appears to be an abundant candidate allelecapable of directly influencing inter-individual variation in behavior,susceptibility to mental disorder, and response to drug treatmentincluding atypical antipsychotic and some antidepressant drugs that arepotent HTR2C inverse agonists or antagonists. A recent study [41]further reported that despite prolonged pre-exposure to serotonincausing equally rapid and strong desensitization of both isoreceptors, agreater cell surface expression and more rapid resensitization followedby SB206553, a mixed antagonist for HTR2B and HTR2C, in Ser-receptorswas observed and might be therapeutically relevant for drugs exhibitinginverse agonist properties at HTR2C. It may be concluded that prolongedexposure of both HTR2C isoreceptors to an inverse agonist increasesreceptor responsiveness to endogenous serotonin or other HTR2C agonists.This enhanced responsiveness occurs earlier in cells (and presumablyalso in individuals) expressing Ser than in those expression Cys.

Whether in vitro findings can be transferable to intact nervous systemis still debatable. Mickey et al. reported that dopaminergic circuitryis more sensitive to pain stress in Ser23 carriers during a PET scanwith a displaceable D2/D3 receptor radiotracer[62]. Greater dopaminerelease in the nucleus accumbens, caudate nucleus, and putamen wasobserved in the Ser23 carriers during pain, suggesting mesoaccumbalstress sensitivity may mediate the effects of HTR2C variation on therisk of neuropsychiatric disorders.

Another explanation for the discrepancies of HTR2C association withsymptom improvement, is mRNA editing[61]. This may help to explain whysome functional variants play more important role inantipsychotic-induced weight gain but others may be more relevant totreatment response. HTR2C receptors are subject to an extraordinarydegree of region-specific RNA editing. This leads to multiple receptorisoforms with dramatically different constitutive activity, expressionpatterns, and efficacy of APDs[63-65]. Altered editing patterns of theHTR2C receptor has been observed in the prefrontal cortex of suicidevictims in a gender-specific pattern[65,66]. There is conflictingevidence for reduced RNA editing, leading to increased expression of theunedited firm of HTR2C in schizophrenic patients[67]. Further study isneeded to determine that HTR2C RNA editing may be involved in themaintenance of appropriate serotonergic neurotransmission and influencetreatment response in psychiatric disorders. Some limited negativefindings have been reported on the relationship between −759C/T orCys23Ser and RNA editing level in brain[68,69]. Although it is stillunclear whether HTR2C polymorphisms have any impact on theregion-specific RNA editing, the present results warrant future studiesaddressing the mechanisms by which the HTR2C polymorphisms, either aloneor in conjunction with other markers, as well as post-transcriptionalmodification, are implicated, in specific features of schizophrenia,such as positive/negative symptoms, and the therapeutic effect of APDs.More studies are needed to elucidate the role of HTR2C geneticpolymorphisms in treatment response with regard to specific types ofsymptom improvement in different ethnic population, well-balanced gender(analyzed separately), larger sample size, and single APD or APDs withsimilar pharmacology.

Supplemental Information

Clinical Evaluation of Treatment Response

The majority of these patients were initially hospitalized for an acuteexacerbation of symptoms or because of failure to respond adequately toconventional or atypical antipsychotic drugs between 1995 and 2010,leading to 118 patients (69%) with initial diagnosis oftreatment-resistant schizophrenia (TRS) as defined, by Kane, et al[48].Demographic information is provided in Table 1A and Table 1B separatedby gender. Over 75% of patients were not medicated or had a drug freeperiod of 3-10 days prior to baseline assessment. Patients wereinterviewed by trained raters using the Schedule for Affective Disordersand Schizophrenia[70] to establish diagnosis. This was integrated withall available data to make the final diagnosis by consensus according tothe Diagnostic and Statistical Manual of Mental Disorders, third edition(DSM-III) criteria at discharge. A review of these diagnoses indicatesall patients meet DSM-IV criteria for schizophrenia or schizoaffectivedisorder. For the purpose of this report, data from patients with eitherdiagnosis are combined. Brief Psychiatric Rating Scale (BPRS; itemsrated 0-6[59] was used to assess the severity of psychopathology. BPRSpositive symptom subscale includes suspiciousness, hallucinatorybehavior, unusual thought content, and conceptual disorganization. In afactor analysis of BPRS ratings in 572 schizophrenia patient, includingthe subjects in this study, conceptual disorganization did not clusterwith suspiciousness, hallucinatory behavior, and unusual thought content(Jayathilake et al unpublished data). Similar findings have beenreported by others [71], suggesting conceptual disorganization is weaklyconnected to the positive symptom domain. We, therefore, excludedconceptual disorganization in the analyses of positive symptoms whichfollow. The remaining three items, suspiciousness, hallucinatorybehavior, and unusual thought content are referred to as the positive3-item. BPRS negative subscale is comprised of three items: emotionalwithdrawal, motor retardation and blunted affect. The anxiety-depressionsubscale consists of three items as well: anxiety, guilty feelings, anddepressive mood. Categorical treatment response was evaluated at 6 weekand 6 months, using the criteria based upon Kane et al, [48]. Areduction of >20% in BPRS total or subscale scores was considered asresponder. In cases where patients were very close to the operationalcriteria for response (>15% or <25%), a reduction of at least onecategory on the Clinical Global Impressions (CGI) scale was consideredin order to enhance the definition of response.

After a description of the study, written informed consent was obtainedfrom every subject. All patients provided written informed consent toremain drug, free during the assessment. The drug free period wasterminated if patient well-being required it.

Some were not receiving psychotropic drugs prior to admission because ofnon-compliance. The study protocol was approved by institutional ethicscommittees and was performed in accordance with the ethical standardslaid down in the 1964 Declaration of Helsinki.

Meta-Analysis

In order to review and elucidate the general relationship between HTR2Cpolymorphisms and drug response to clozapine, a Pubmed search wasconducted using Medline databases from 1966 to February, 2014. Thefollowing, combination of search terms, “antipsychotic agents”[MeSHTerms] AND 5-HTR2C[All Fields] OR HTR2C[All Fields] AND “humans”[MeSHTerms], help to identified 217 abstracts.

There are 11 English-language citations which were extracted because ofthe topic on drug response in schizophrenia with reported genotype dataon HTR2C polymorphisms. The references listed from obtained articleswere also searched to identify further relevant citations. There was alack of study of rs6318 in East Asian population and rs3813929 inAfrican-American population due to very low MAF (<5%, Table 9). Asshowed in Table 2, the following information was collected from thesepapers: names of first authors with year of publication, number ofsubject (separated by gender), percentage of patients on clozapine orother APDs, ethnicity (European, African-American, East Asian), studyduration, observed genetic variants in HTR2C, Phenotype observed (binaryand/or quantitative), Responder/No responder Ratio, Statisticalanalysis, and summary of the results.

Those full-text articles were then scrutinized by two authors. J Li andH Y Meltzer, to determine eligibility for inclusion in themeta-analysis. Only rs6318 for which more than two studies have beenpublished. Finally, six studies, including ours, with accessiblegenotyping data for rs6318 and binary outcome for symptom improvement,were included. We did not stratify the patients for each study based onthe gender or ethnicity. Only two studies (ours and Masellis') recruiteda small portion of African-American subjects. This is an extension of apreviously reported meta-analysis on the same topic[57]. Although over50% of the subjects received clozapine or olanzapine treatment in eachstudy except Vehof's, the definition of drug responder varied. We didnot include a candidate-gene study of SNP array data generated from theCATIE sample[72] because clozapine was not the observed APD and thetrait for treatment response is quantitative but not binary (Table 2).There is no significant deviation from HWE (p>0.001) for rs6318 in eachstudies. In order to reduce the heterogeneity among these studies andavoid the publication bias, we included all patients (male and female)and only improvement in positive symptoms was considered in our study inorder to match another study [56]. Heterogeneity among the studies wasassessed by means of the I² inconsistency test and Cochran's Qstatistics under a null hypothesis test in which p<0.05.

Number of studies combined: k=6

OR 95%-CI z p.value

Fixed effect model 2.0038 [1.3801; 2.9093]3.6535 0.0003

Random effects model 1.9437 [1.2655; 2.9851]3.0357 0.0024

Quantifying heterogeneity:

tau{circumflex over ( )}2=0.0491; H=1.1 [1; 1.62]; I{circumflex over( )}2=17% [0%; 61.8%]

Test of heterogeneity: Q d.f. p.value

6.03 5 0.3037

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It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein. The terms and expressions whichhave been employed are used as terms of description and not oflimitation, and there is no intention in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention. Thus, itshould be understood that although the present invention has beenillustrated by specific embodiments and optional features, modificationand/or variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention.

Citations to a number of patent and non-patent references may be madeherein. Any cited references are incorporated by reference herein intheir entireties. In the event that there is an inconsistency between adefinition of a term in the specification as compared to a definition ofthe term in a cited reference, the term should be interpreted based onthe definition in the specification.

We claim:
 1. A method comprising: (a) determining that a subject havingschizophrenia and exhibiting negative symptoms of schizophreniacomprises a combination of HTR2C polymorphic alleles by detecting in anucleic acid sample from the subject the combination of HTR2Cpolymorphic alleles, wherein the negative symptoms of schizophrenia areselected from emotional withdrawal, motor retardation, and bluntedeffect, wherein the combination of HTR2C polymorphic alleles comprises apolymorphic allele resulting in a Cys23Ser amino acid substitution, anda C-allele of the rs3813929 (−759C/T) polymorphism, and whereindetecting comprises amplifying at least a portion of the HTR2C genecomprising the combination of HTR2C polymorphic alleles from the nucleicacid sample and sequencing the amplified portion; and (b) afterdetermining that the subject having schizophrenia and exhibitingnegative symptoms of schizophrenia comprises the combination of HTR2Cpolymorphism alleles by detecting the combination of HTR2C polymorphicalleles in the nucleic acid sample from the subject, administering tothe subject an atypical antipsychotic drug.
 2. The method of claim 1,wherein detecting comprises detecting whether the nucleic acid sample ishomozygous for a HTR2C polymorphic allele of the combination of HTR2Cpolymorphic alleles.
 3. The method of claim 1, wherein detectingcomprises detecting whether the nucleic acid sample is heterozygous fora HTR2C polymorphic allele of the combination of HTR2C polymorphicalleles.
 4. The method of claim 1, wherein the nucleic acid sample isobtained from blood or a blood product.
 5. The method of claim 1,wherein the atypical antipsychotic drug is an HTR2C inverse agonist. 6.The method of claim 1, wherein the antipsychotic drug is selected fromclozapine, melperone, risperidone, and olanzapine.
 7. A methodcomprising: (a) determining that a subject having schizophrenia andexhibiting negative symptoms of schizophrenia comprises a combination ofHTR2C polymorphic alleles by detecting in a nucleic acid sample from thesubject the combination of HTR2C polymorphic alleles, wherein thenegative symptoms of schizophrenia are selected from emotionalwithdrawal, motor retardation, and blunted effect, wherein thecombination of HTR2C polymorphic alleles comprises a polymorphic alleleresulting in a Cys23Ser amino acid substitution, and a C-allele of thers3813929 (−759C/T) polymorphism, and wherein detecting comprisesamplifying at least a portion of the HTR2C gene comprising thecombination of HTR2C polymorphic alleles from the nucleic acid sampleand contacting the amplified portion with one or more nucleic acidprobes that hybridize specifically to nucleic acid comprising thecombination of HTR2C polymorphic alleles: and (b) after determining thatthe subject having schizophrenia and exhibiting negative symptoms ofschizophrenia comprises the combination of HTR2C polymorphism alleles bydetecting the combination of HTR2C polymorphic alleles in the nucleicacid sample from the subject, administering to the subject an atypicalantipsychotic drug.
 8. The method of claim 7, wherein the antipsychoticdrug is selected from clozapine, melperone, risperidone, and olanzapine.9. The method of claim 7, wherein the antipsychotic drug is an HTR2Cinverse agonist.
 10. The method of claim 7, wherein detecting comprisesdetecting whether the nucleic acid sample is homozygous for a HTR2Cpolymorphic allele of the combination of HTR2C polymorphic alleles. 11.The method of claim 7, wherein detecting comprises detecting whether thenucleic acid sample is heterozygous for a HTR2C polymorphic allele ofthe combination of HTR2C polymorphic alleles.
 12. The method of claim 7,wherein the nucleic acid sample is obtained from blood or a bloodproduct.